Complexes possessing ortho ligating functionality

ABSTRACT

A group of functionalized amine chelants having ortho ligating functionality that form complexes with rare-earth type metal ions are disclosed. The chelants possess the general formula ##STR1## wherein R &#39;1 , R &#39;2 , R &#39;3 , R &#39;4 , R &#39;5  and R &#39;6 , Z&#39; and X are all defined in the specification. In addition certain of the chelant-radionuclide complexes can be effectively employed in compositions useful as therapeutic and/or diagnostic agents for calcific tumors and/or relief of bone pain.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of copending applicationU.S.S.N. 421,452, filed Oct. 13, 1989, which is a continuation-in-partof application U.S.S.N. 265,158, filed Oct. 31, 1988, now abanboned.

1. Field of the Invention

The present invention concerns chelants passessing ortho ligatingfuctionality, complexes and conjugates thereof, processes for theirpreparation, formulations for their use and methods for their use incancer diagnostics and/or therapy.

2. Background of the Invention

Functionalized chelants, or bifunctional coordinators, are known to becapable of being covalently attached to an antibody having specificityfor cancer or tumor cell epitopes or antigens. Radionuclide complexes ofsuch antibody/chelant conjugates are useful in diagnostic and/ortherapeutic applications as a means of conveying the radionuclide to acancer or tumor cell. See, for example, Meares et al., Anal. Biochem.142, 68-78, ( 1984 ); and Krejcarek et al., Biochem. and Biophys. Res.Comm. 77, 581-585 ( 1977 ).

Aminocarboxylic acid chelating agents have been known and studied in theliterature for several years. Typical of the aminocarboxylic acids arenitrilotri-acetic acid (NTA), ethylenediaminetetraacetic acid (EDTA),hydroxyethylethylenediaminetriacetic acid (HEDTA),diethylenetriaminepentaacetic acid (DTPA) andtrans-1,2-diaminocyclohexanetetraacetic acid (CDTA). Numerousbifunctional chelating agents based on aminocarboxylic acids have beenproposed and prepared. For example the cyclic dianhydride of DTPA(Hnatowich et.al. Science, 220, 613-615, 1983; U.S. Pat. No. 4,479,930)and mixed carboxycarbonic anhydrides of DTPA (Gansow, U.S. Pat. Nos.4,454,106 and 4,472,509; Krejcarek et al., Biochem. and Biophys. Res.Comm. 77, 581-585, 1977) have been reported in the literature. When theanhydrides are coupled to proteins the coupling proceeds via formationof an amide bond thus leaving four of the original five carboxymethylgroups on the diethylenetriamine (DETA) backbone (Hnatowich et al., Int.J. Appl. Isot. 33, 327-332, 1982). In addition, U.S. Pat. Nos. 4,432,907and 4,352,751 disclose bifunctional chelating agents useful for bindingmetal ions to "organic species such as organic target molecules orantibodies." As in the above, coupling is obtained via an amide groupthrough the utilization of diaminotetraacetic acid dianhydrides.Examples of anhydrides include dianhydrides of EDTA, CDTA,propylenediaminetetraacetic acid and phenylene 1,2-diaminetetraaceticacid. A recent U.S. Pat. No. 4,647,447 discloses several complex saltsformed from the anion of a complexing acid for use in various diagnostictechniques. Conjugation via a carboxyl group of the complexing acid istaught which gives a linkage through an amide bond.

Another class of bifunctional chelating agent based on aminocarboxylicacid functionality is also well documented in the literature. Thus,Sundberg et al. in the J. of Med. Chem. 17(12), 1304 (1974) disclosesbifunctional analogs of EDTA. Representative of these compounds are1-(p-nitrophenyl)ethylenediaminetetraacetic acid,1-(p-aminophenyl)ethylenediaminetetraacetic acid, and1-(p-benzenediazonium)ethylenediaminetetraacetic acid. Coupling toproteins through the para-substituent and the binding of radio-activemetal ions to the chelating group is discussed. The compounds are alsodisclosed in Biochem. Biophys. Res. Comm. 75(1), 149 (1977) and in U.S.Pat. Nos. 3,994,966 and 4,043,998. It is important to note thatattachment of the aromatic group to the EDTA structure is through acarbon of the ethylenediamine backbone. Optically active bifunctionalchelating agents based on EDTA, HEDTA and DTPA are disclosed in U.S.Pat. No. 4,622,420. Also in this reference the attachment of theaminocarboxylic acid functionality to the rest of the bifunctionalchelating molecule is through a carbon of the ethyleneamine backbone. Inthese compounds an alkylene group links the aromatic group (whichcontains the functionality needed for attachment to the protein) to thecarbon of the polyamine which contains the chelating functionality.Other references to such compounds include Brechbiel et al. Inorg. Chem.25, 2772-2781 (1986), U.S. Pat. No. 4,647,447 and a published PCTapplication having International Publication Number WO 86/06384. Morerecently, certain macrocyclic bifunctional chelating agents and the useof their copper chelate conjugates for diagnostic or therapeuticapplications have been disclosed in U.S. Pat. No. 4,678,667. Attachmentof the aminocarboxylic acid functionality to the rest of thebifunctional chelating molecule is through a ring carbon of the cyclicpolyamine backbone. Thus, a linker, attached at one end to a ring carbonof the cyclic polyamine, is also attached at its other end to afunctional group capable of reacting with the protein.

Another class of bifunctional chelating agent, also worthy of note,consists of compounds wherein the chelating moiety, i.e. theaminocarboxylic acid, of the molecule is attached through a nitrogen tothe functional group of the molecule containing the moiety capable ofreacting with the protein. As an example Mikola et al. in a publishedPCT application (International Publication Number WO 84/03698, publishedSep. 27, 1984) disclose a bifunctional chelating agent prepared byreacting p-nitrobenzylbromide with DETA followed by reaction withbromoacetic acid to make the aminocarboxylic acid. The nitro group isreduced to the corresponding amine group and is then converted to theisothiocyanate group by reaction with thiophosgene. These compounds arebifunctional chelating agents which can be conjugated to bio-organicmolecules for use as diagnostic agents capable of chelating lanthanides.Since attachment of the linker portion of the molecule is through one ofthe nitrogens of the aminocarboxylic acid, then one potentialaminocarboxyl group is lost for chelation. Thus, a DETA-basedbifunctional chelant containing four (not five) acid groups is prepared.In this respect this class of bifunctional chelant is similar to thosewhere attachment to the protein is through an amide group withsubsequent loss of a carboxyl chelating group.

In the J. Radioanalytical Chem. 57(12), 553-564 (1980), Paik et al.disclose the use of p-nitrobenzylbromide in a reaction with a "blocked"diethylenetriamine, i.e. bis-(2-phthalimidoethyl)amine followed bydeblocking procedures and carboxymethylation using chloroacetic acid, togive N'-p-nitrobenzyldiethylenetriamine N,N,N",N"-tetraacetic acid.Again, since the attachment is through a nitrogen, a tetraacetic acidderivative is obtained. Conjugation of the bifunctional chelating agentand chelation with indium is discussed. Substitution on the nitrogenatom is also taught by Eckelman et al. in the J. Pharm. Sci. 64(4),(1975) by reacting amines such as "ethylenediamine or diethylenetriaminewith the appropriate alkyl bromide before carboxymethylation." Thecompounds are proposed as potential radiopharmaceutical imaging agents.

Recently Carney, Rogers, and Johnson disclosed (3rd. InternationalConference on Monoclonal Antibodies; San Diego, Calif.--Feb. 4-6, 1988)abstracts entitled "Absence of Intrinsically Higher Tissue Uptake fromIndium-111 Labeled Antibodies: Co-administration of Indium-111 andIodine-125 Labeled B72.3 in a Nude Mouse Model" and "Influence ofChelator Denticity on the Biodistribution of Indium-111 Labeled B72.3Immunoconjugates in Nude Mice". The biodistribution of indium-111complexed with an EDTA and DTPA bifunctional chelating agent isdisclosed. Attachment of the aromatic ring to the EDTA/DTPA moleties isthrough an acetate radical. Previously Hunt et al. in U.S. Pat. Nos.4,088,747 and 4,091,088 (1978) disclosed ethylenediaminediacetic acid(EDDA) based chelating agents wherein attachment of an aromatic ring tothe EDDA moiety is through the alkylene or acetate radical. Thecompounds are taught to be useful as chelates for studying hepatobillaryfunction. The preferred metal is technetium-99m. Indium-111 and indium113 are also taught as useful radionuclides for imaging.

Martell et al. in the Inorganica Chemica Acta 138, 215-230 (1987)disclose an iron chelating agent for treating Cooley's anemia. Theligands used were analogs of EDTA with amino and carboxylate donorgroups, or having additional donor groups present like phenolic orphenolic groups substituted on pyridine rings; aminophosphonic acid orester groups with additional phenolate and amino donors; macrocyclicpolyamines having carboxylate and/or phenolate donor groups;trishydroxamic acids; triscatechols; and multidentate ligands withcoordinating amide groups.

The development of bone metastases is a common and often catastrophicevent for a cancer patient. The pain, pathological fractures, frequentneurological deficits and forced immobility caused by these metastaticlesions significantly decrease the quality of life for the cancerpatient. The number of patients that contract metastatic disease islarge since nearly 50% of all patients who contract breast, lung orprostate carcinoma will eventually develop bone metastases. Bonemetastases are also seen in patients with carcinoma of the kidney,thyroid, bladder, cervix and other tumors, and collectively, theserepresent less than 20% of patients who develop bone metastases.Metastatic bone cancer is rarely life threatening and occasionallypatients live for years following the discovery of the bone lesions.Initially, treatment goals center on relieving pain, reducingrequirements for narcotic medication and increasing ambulation. Clearly,it is hoped that some of the cancers can be cured.

The use of radionuclides for treatment of cancer metastatic to the bonedates back to the early 1950's. It has been proposed to inject aradioactive particle-emitting nuclide in a suitable form for thetreatment of calcific lesions. It is desirable that such nuclides beconcentrated in the area of the bone lesion with minimal amountsreaching the soft tissue and normal bone. Radioactive phosphorus (P-32and P-33) compounds have been proposed, but the nuclear andbiolocalization properties limit the utility of these compounds.[Kaplan, E., et al., J. Nuc. Med. 1(1), 1, (1960); (U.S. Pat. No.3,965,254)].

Another attempt to treat bone cancer has been made using phosphoruscompounds containing a boron residue. The compounds were injected intothe body (intravenously) and accumulated in the skeletal system. Thetreatment area was then irradiated with neutrons in order to activatethe boron and give a therapeutic radiation dose. (U.S. Pat. No.4,399,817).

In the above mentioned procedures, it is not possible to givetherapeutic doses to the tumor without substantial damage to normaltissues. In many cases, especially for metastic bone lesions, the tumorhas spread throughout the skeletal system and amputation or irradiationis not practical. (Seminars in Nuclear Medicine IX(2), April, 1979).

The use of Re-186 complexed with a diphosphonate has also been proposed.[Mathieu, L. et al., [Int. J. App. Rad. & Isot. 30, 725-727 (1979);Weinenger, J., Ketring, A. R., et al., J. Nuc. Med. 24(5), 125, (1983)].However, the preparation and purification needed for this complex limitsits utility and wide application.

Strontium-89 has also been proposed for patients with metastic bonelesions. However, the long half-life (50.4 days), high blood levels andlow lesion to normal bone ratios can be disadvantageous. [Firusian, N.,Mellin, P., Schmidt, C. G., The Journal of Urology, 116, 764, (1976);Schmidt, C. G., Firusian, N., Int. J. Clin. Pharmacol., 93, 199-205,(1974)].

A palliative treatment of bone metastases has been reported whichemployed I-131 labelledα-amino-(3-iodo-4-hydroxybenzylidene)diphosphonate [Eisenhut, M., J.Nuc. Med. 25(12), 1356-1361, (1984)]. The use of radioiodine as atherapeutic radionuclide is less than desirable due to the well knowntendency of iodine to localize in the thyroid. Eisenhut lists iodide asone of the possible metabolites of this compound. In addition, any I-131left over from the iodination reaction and not separated in the washingprocedure also constitutes a threat to the thyroid.

Aminocarboxylic acids are known to chelate metal ions. Particularlystable chelates are formed with metals from the alkaline earth andtransition metal series.

O'Mara et al. (J. Nuc. Med. 10, 49-51, 1969) have prepared rare earthcomplexes of aminocarboxylic acids at chelant to metal ratios of 10:1.They find good skeletal properties and propose their use as diagnosticskeletal agents. In addition to high bone uptake, high amounts ofradiation were observed in muscle and/or liver. Of the rare earthnuclides evaluated Sm-153 and Er-171 were indicated as having the mostsuitable characteristics for imaging in humans. The utility of theseagents for therapy, however, is not suggested.

Rosoff, B. et al., Int. J. App. Rad. and Lot. 14, 129-135 (1963),disclose complexes of EDTA and NTA with certain radionuclides, namelySc-46, Y-91, La-140 and Sm-153. The relationship of the stabilityconstant of these complexes to urinary excretion is shown. Chelant tometal molar ratios of 5:1 were employed and high concentrations ofradioactivity were observed in the liver, spleen, kidney, lung and bone.

SUMMARY OF THE INVENTION

The present invention is directed to novel chelants possessing ortholigating functionality, which chelant forms complexes with metals,especially "radioactive" metals having rare earth-type chemistry.Preferred radioactive metals include samarium-153 (¹⁵³ Sm), holmium-166(¹⁶⁶ Ho), yttrium-90 (⁹⁰ y), promethium-149 (¹⁴⁹ Pm), gadolinium-159(¹⁵⁹ Gd), lanthanum-140 (¹⁴⁰ La), lutetium-177 (¹⁷⁷ Lu), ytterbium-175(¹⁷⁵ Yb), scandium-47 (⁴⁷ Sc) and praseodymium-142 (¹⁴² Pr). Thecomplexes so formed can be used by themselves or can be attached to anantibody or fragment thereof and used for therapeutic and/or diagnosticpurposes. The complexes and/or conjugates can be formulated for in vivoor in vitro uses. Preferred uses of the formulated conjugates is thetreatment of cancer in animals, especially humans.

In addition certain of the chelant-radionuclide complexes can beeffectively employed in compositions useful as therapeutic and/ordiagnostic agents for calcific tumors and/or in compositions useful astherapeutic agents for the relief of bone pain.

More specifically, the present invention is directed to compounds havingthe formula:wherein: ##STR2##

Z is an electrophilic or nucleophilic moiety which allows for covalentattachment to an antibody or fragment thereof or a synthetic linkerwhich does not interfere with the formation of complexation with aradionuclide and which can be attached to an antibody or fragmentthereof;

X is hydrogen, C₁ -C₃ alkyl or CR₃ R₄ CO₂ H;

R₁, R₂, R₃ and R₄ each are independently hydrogen, hydroxy, CO₂ H or aC₁ -C₃ alkyl group;

R₅ is hydrogen or (CR₁ R₂)_(n) CR₃ R₄ B';

B represents a linear or branched polyalkylene polyamine where at leastone of the amine hydrogens has been substituted with a CR₃ R₄ CO₂ Hgroup;

B' represents a linear or branched amine or polyalkylene polyamine whereat least one of the amine hydrogens has been substituted with a CR₃ R₄CO₂ H group;

n is 0 or 1; or

a pharmaceutically acceptable salt thereof.

Also included within the scope of the present invention are compoundshaving a chelant posssessing ortho ligating functionality having theformula ##STR3## wherein:

X is hydrogen, C₁ -C₃ alkyl or CR₃ R₄ CO₂ H;

R₁, R₂, R₃ and R₄ each are independently hydrogen, hydroxy, CO₂ H or aC₁ -C₃ alkyl group;

R₅ is hydrogen or (CR₁ R₂)_(n) CR₃ R₄ B';

B represents a linear or branched polyalkylene polyamine where at leastone of the amine hydrogens has been substituted with a CR₃ R₄ CO₂ Hgroup;

B' represents a linear or branched amine or polyalkylene polyamine whereat least one of the amine hydrogens has been substituted with a CR₃ R₄CO₂ H group;

n is 0 or 1; or

a pharmaceutically acceptable salt thereof.

It is preferred that the carboxyl group (when present) be attached tothe first or second carbon from the nitrogen of the B group, i.e. thecarbon α or β to the nitrogen in the chelant moiety. Preferred compoundsof formula I are those where n is 0; or R₁, R₂, R₃ and R₄ are eachhydrogen; or n is 0 and one of R₃ or R₄ is hydrogen and the other isCOOH; or X is hydrogen. When the chelants are to be used as bifunctionalchelating agents, then Z is preferably amino, isothiocyanato,semicarbazide, thiosemicarbazide, carboxyl, bromoacetamido or maleimido.

Additionally, the present invention is directed to compounds having theformula: ##STR4##

wherein Z' is hydrogen, NH₂, NO₂, NHC(O)CH₃ or N(R')₂, where R' ishydrogen or C₁ -C₃ alkyl;

X is hydrogen, C₁ -C₃ alkyl or CR₃ R₄ COOH;

R'₁ is hydrogen or COOH;

R'₃, R'₄ and R'₅ are independently hydrogen or CR₃ R₄ COOH, with theproviso that at least one of R'₁, R'₃, R'₄ and R'₅ is hydrogen; or

a pharmaceutically acceptable salt thereof.

Furthermore, the present invention is directed to compounds having theformula: ##STR5##

wherein Z' is selected from the group consisting of hydrogen, NH₂, NO₂,NHC(O)CH₃ or N(R')₂, where R' is hydrogen and C₁ -C₃ alkyl;

X is selected from the group consisting of hydrogen, C₁ -C₃ alkyl andCR₃ R₄ COOH;

R₃ and R₄ are independently selected from the group consisting ofhydrogen, C₁ -C₃ alkyl and COOH;

R'₁ and R'₂ each are independently selected from the group consisting ofhydrogen and COOH, with the proviso that at least one is COOH;

R'₃, R'4, R'₅ and R'₆ are independently selected from the groupconsisting of hydrogen and CR₃ R₄ COOH, with the proviso that at leastthree are CR₃ R₄ COOH; or

a pharmaceutically acceptable salt thereof.

Also included in the scope of this invention are complexes andconjugates and methods of use of the compounds of Formula III. Suchcomplexes comprise the compound complexed with a radionuclide metal ionselected from the group consisting of ¹⁵³ Sm, ¹⁶⁶ Ho, ⁹⁰ Y, ¹⁴⁹ pm, ¹⁵⁹Gd, ¹⁴⁰ La, ¹⁷⁷ Lu, ¹⁷⁵ Yb, ⁴⁷ Sc and ¹⁴² Pr.

The present invention is also directed to radioactive metal ioncomplexes, especially radioactive rare-earth type metal ion complexes,and to conjugates formed with the aforementioned complexes and antibodyor antibody fragments. In addition, the present invention also includesformulations having the chelant-radionuclide complexes and/or theconjugates of the invention and a pharmaceutically acceptable carrier.Typically the pharmaceutically acceptable carrier in these formulationsis in liquid form. The invention also includes a method for thediagnosis or treatment of a disease state, especially cancer, in amammal by the administration to the mammal an effective amount of theformulation.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the following indicated terms have these meanings: withrespect to the definition of Z; "electrophilic" moieties include, butare not limited to, isothiocyanate, bromoacetamide, maleimide,imidoester, thiophthalimide, N-hydroxysuccinimyl ester, pyridyldisulfide and phenyl azide; suitable "nucleophilic" moleties include,but are not limited to, carboxyl, amino, acyl hydrazide, semicarbazide,and thiosemicarbazide; "synthetic linkers" include any synthetic organicor inorganic linkers which are capable of being covalently attached toan antibody or antibody fragment, preferred synthetic linkers arebiodegradable synthetic linkers which are stable in the serum of apatient but which have a potential for enzymatic cleavage within anorgan of clearance for the radioisotope, for example biodegradablepeptides or peptide containing groups. Of the electrophilic moietiesisothiocyanate, bromoacetamide and maleimide are preferred, especiallypreferred is isothiocyanate; and of the nucleophilic moleties amino,carboxyl, semicarbazide and thiosemicarbazide are preferred, especiallypreferred are amino and carboxyl. It is desirable that the nature and/orposition of Z be such that it does not appreciably interfere with thechelation reaction. Z can also be a non-reactive moiety such as H, NO₂,NHC(0)CH₃, NR'₂ (where R' is H or C₁ -C₃ alkyl) when the end use doesnot involve attachment of the chelate to a protein.

The term "C₁ -C₃ " alkyl includes methyl, ethyl, n-propyl and isopropyl.

The terms "linear or branched amine or polyalkylene amine" mean straightor branched chain alkyl moieties that contain at least one, and usuallymore than one, nitrogen atom.

As used herein, the term "mammal" means animals that nourish their youngwith milk secreted by mammary glands, preferably warm blooded mammals,more preferably humans.

"Antibody" refers to any polyclonal, monoclonal, chimeric antibody orheteroantibody, preferably a monoclonal antibody; "antibody fragment"includes Fab fragments and F(ab')₂ fragments, and any portion of anantibody having specificity toward a desired epitope or epitopes. Whenusing the term "radioactive metal chelate/antibody conjugate" or"conjugate" the "antibody" is meant to include whole antibodies and/orantibody fragments, including semisynthetic or genetically engineeredvariants thereof. Preferred antibodies are CC-49 and antibody fragmentssuch as Fab and F(ab')₂. Other possible antibodies are CC-83 and B72.3.The hybridoma cell line B72.3 is deposited American Type CultureCollection (ATCC), having the accession number ATCC HB 8108. The othermurine monoclonal antibodies bind to epitopes of TAG-72, a tumorassociated antigen.

As used herein, "radioactive metal complex" or "complex" refers to acomplex of the compound of the invention, e.g. formula I, complexed witha rare-earth type metal ion, especially a radioactive rare-earth typemetal ion, where at least one metal atom is chelated or sequestered;"radioactive metal ion chelate/antibody conjugate" or "radioactive metalion conjugate" refers to a radioactive metal ion conjugate that iscovalently attached to an antibody or antibody fragment; "radioactive"when used in conjunction with the word "metal ion" refers to one or moreisotopes of the rare-earth type elements that emit particles and/orphotons, such as ¹⁵³ Sm, ¹⁶⁶ Ho, ⁹⁰ Y, ¹⁴⁹ pm, ¹⁵⁹ Gd, ¹⁴⁰ La, ¹⁷⁷ Lu,¹⁷⁵ Yb, ⁴⁷ Sc and ¹⁴² Pr. The terms "bifunctional coordinator""bifunctional chelating agent" and "functionalized chelant" are usedinterchangeably and refer to compounds that have a chelant moietycapable of chelating a metal ion and a linker/spacer moiety covalentlybonded to the chelant moiety that is capable of serving as a means tocovalently attach to an antibody or antibody fragment.

As used herein, "pharmaceutically acceptable salt" means any salt of acompound of formula (I) which is sufficiently non-toxic to be useful intherapy or diagnosis of mammals. Thus, the salts are useful inaccordance with this invention. Representative of those salts formed bystandard reactions from both organic and inorganic sources include, forexample, sulfuric, hydrochloric, phosphoric, acetic, succinic, citric,lactic, maleic, fumaric, palmitic, cholic, pamoic, mucic, glutamic,d-camphoric, glutaric, glycolic, phthalic, tartaric, formic, lauric,steric, salicylic, methanesulfonic, benzenesulfonic, sorbic, picric,benzoic, cinnamic acids and other suitable acids. Also included aresalts formed by standard reactions from both organic and inorganicsources such as ammonium, alkali metal ions, alkaline earth metal ions,and other similar ions. Particularly preferred are the salts of thecompounds of formula (I) where the salt is potassium, sodium, ammonium,or mixtures thereof.

The bifunctional chelating agents described herein (represented byformula I) can be used to chelate or sequester the rare-earth type metalions, particularly radioactive rare-earth type metal ions, so as to formmetal ion chelates (also referred to herein as "complexes"). Thecomplexes, because of the presence of the functionalizing moiety(represented by "Z" in formula I), can be attached to functionalizedsupports, such as functionalized polymeric supports, or preferablycovalently attached to antibodies or antibody fragments. Thus thecomplexes described herein may be covalently attached to an antibody orantibody fragment and are referred to herein as "conjugates".

The antibodies or antibody fragments which may be used in the conjugatesdescribed herein can be prepared by techniques well known in the art.Highly specific monoclonal antibodies can be produced by hybridizationtechniques well known in the art, see for example, Kohler and Milstein[Nature 256,495-497 (1975); and Eur. J. Immunol. 6, 511-519 (1976)].Such antibodies normally have a highly specific reactivity. In theantibody targeted radioactive metal ion conjugates, antibodies directedagainst any desired antigen or hapten may be used. Preferably theantibodies which are used in the radioactive metal ion conjugates aremonoclonal antibodies, or fragments thereof having high specificity fora desired epitope(s). Antibodies used in the present invention may bedirected against, for example, tumors, bacteria, fungi, viruses,parasites, mycoplasma, differentiation and other cell membrane antigens,pathogen surface antigens, toxins, enzymes, allergens, drugs and anybiologically active molecules. Some examples of antibodies or antibodiyfragraments are CC-11, CC-15, CC-30, CC-46, CC-49 F(ab')₂, CC-49, CC-83,CC-83 F(ab')₂, CC-92 and B72.3. [See D. Colcher et al., Cancer Res. 48,4597-4603 (Aug. 15, 1988) for CC-49, CC-83 and B72.3 antibodies.] Thefollowing CC antibodies have been deposited in ATCC as follows: CC-11 asHB 9455; CC-15 as HB 9460; CC-30 as HB 9457; CC-46 as HB 9458; CC-49 asHB 9459; CC-83 as HB 9453; and CC-92 as HB 9454. B72.3 has beendeposited in ATCC as HB 8108. A more complete list of antigens can befound in U.S. Pat. No. 4,193,983. The radioactive metal ionchelate/antibody conjugates of the present invention are particularlypreferred for the diagnosis and treatment of various cancers.

The preferred rare-earth type (lanthanide or pseudo-lanthanide)complexes of the present invention are represented by the formula:

    C[Ln(BFC)]

wherein: Ln is a rare-earth metal (lanthanide) ion, such as Ce³⁺ Pr³⁺Nd³⁺ Pm³⁺ Sm³⁺ Eu³⁺ Gd³⁺ Tb³⁺ Dy³⁺ Ho³⁺ Er³⁺ Tm³⁺ Yb³⁺ and Lu³⁺, orpseudo-lanthanide metal ion, such as Sc³⁺, Y³⁺ and La³⁺ ; BFC representsa bifunctional chelant; and C represents a pharmaceutically acceptableion or group of ions of sufficient charge to render the entire complexneutral. If the BFC contains four or more negatively charged moieties,then C is a cation or group of cations such as H⁺, Li⁺, Na⁺, K⁺, Rb⁺,Cs⁺, Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺, Ra²⁺, NH₄ ⁺, N(CH₃)⁴ ⁺, N(C₂ HS)₄ ⁺, N(C₃H₇)₄ ⁺, N(C₄ H₉)₄ ⁺ , As(C₆ H₅)₄ ⁺, [(C₆ H₅)₃ P=]₂ N⁺ and otherprotonated amines. If the BFC N⁺ contains three negatively chargedmoieties, then C is not required. If the BFC contains two negativelycharged moieties, then C is an anion such as F⁻, Cl⁻, Br⁻, I⁻, C10₄ ⁻,BF₄ ⁻, H₂ PO₄ ⁻, HCO₃ ⁻, HCO₂ ⁻, CH₃ SO₃ ⁻, H₃ C-C₆ H₄ -SO₃ ⁻, PF₆ ⁻,CH₃ CO₂ ⁻ and B (C₆ H₅)₄ ⁻.

This invention is used with a physiologically acceptable carrier,excipient or vehicle therefor. The methods for preparing suchformulations are well known. The formulations may be in the form of asuspension, injectable solution or other suitable formulations.Physiologically acceptable suspending media, with or without adjuvants,may be used.

An "effective amount" of the formulation is used for therapy. The dosewill vary depending on the disease being treated. Although in vitrodiagnostics can be performed with the formulations of this invention, invivo diagnostics are also contemplated using formulations of thisinvention. The conjugates and formulations of this invention can also beused in radioimmuno guided surgery (RIGS); however, other metals whichcould be used for this purpose also include ^(99m) Tc, ¹¹¹ In, ^(113n)In, ⁶⁷ Ga and ⁶⁸ Ga.

When the chelant-radionuclide complexes of this invention are to be usedfor the treatment of bone cancer certain criteria must be met. While theproperties of the radionuclide are important, the overall properties ofthe composition containing the radionuclide-chelant complex is thedetermining factor. The disadvantages of any one property may beovercome by the superiority of one or more of the properties of eitherligand or radionuclide and their combination, as employed in thecomposition must be considered in toto.

The following is a discussion of those criteria which must be consideredin choosing any particular combination (i.e., complex) of radionuclideand ligand employed in the compositions of the invention.Radionuclide-chelant complexes, when used in the absence of anappropriate excess of the ligands employed in the invention may not betherapeutically useful or effective.

There is a need, therefore, for compositions possessing the followingcriteria by which it is possible to deliver therapeutic radiation dosesto calcific tumors with minimal doses to soft tissue.

The radionuclide must be delivered preferentially to the bone ratherthan to soft tissue. Most particularly, uptake in either liver or bloodis undesirable.

The radionuclide should be cleared rapidly from non-osseous tissue toavoid unnecessary damage to such tissues, e.g., it should clear rapidlyfrom the blood.

The proposed use for some of the compositions of this invention is thetherapeutic treatment of calcific tumors in animals. As used herein, theterm "calcific tumors" includes primary tumors, where the skeletalsystem is the first site of involvement, and metastatic bone cancerwhere the neoplasm spreads from other primary sites, such as prostateand breast, into the skeletal system. This invention provides a means ofalleviating pain and/or reducing the size of, and/or inhibiting thegrowth and/or spread of, or causing regression of and/or destroying thecalcific tumors by delivering a therapeutic radiation dose.

The composition may be administered as a single dose or as multipledoses over a longer period of time. Delivery of the radionuclide to thetumor must be in sufficient amounts to provide the benefits referred toabove.

Other uses of some of the chelants of the present invention may includethe removal of undesirable metals (i.e. iron) from the body, magneticresonance imaging, attachment to polymeric supports for variouspurposes, e.g. as diagnostic agents, and removal of lanthanide metal orpseudo-lanthanide metal ion by selective extraction. In addition themetal-ligand complexes used to deliver radionuclides to calcific sitesmay have utility for the ablation of bone marrow (i.e. for bone marrowtransplants).

Radionuclides can be produced in several ways. In a nuclear reactor anuclide is bombarded with neutrons to obtain a radionuclide, e.g.

    Sm-152+neutron→Sm-153+gamma

Another method of obtaining radionuclides is to bombard nuclides withparticles produced by a linear accelerator or a cyclotron. Yet anotherway is to isolate the radionuclide from a mixture of fission products.The method of obtaining the nuclides employed in the present inventionis not critical thereto.

The chelating agents disclosed herein can be prepared in ways well knownto the art. Thus, for example, see Chelating Agents and Metal Chelates,Dwyer & Mellor, Academic Press (1964), Chapter 7. See also methods formaking amino acids in Synthetic Production and Utilization of AminoAcids, (edited by Kameko, et al.) John Wiley & Sons (1974).

When Z (in the formula) is chosen to be an electrophilic moiety it canbe prepared by methods known to the art. Such methods may be found inAcc. Chem. Res. 17., 202-209 (1984 ).

Examples of some of the methods which may be used to prepare thechelants of formula I, II or III are:

A) reacting a compound of the formula ##STR6## wherein:

Z is an electrophilic or nucleophilic moiety which allows for covalentattachment to an antibody or fragment thereof or a synthetic linkerwhich does not interfere with the formation of complexation with aradionuclide and which can be attached to an antibody or fragmentthereof;

X is hydrogen;

R₅ is hydrogen or (CR₁ R₂)_(n) CR₃ R₄ T, where R₁, R₂, R₃ and R₄ eachare independently hydrogen, hydroxy, CO₂ H or a C₁ -C₃ alkyl group, n is0 or 1, and T represents a linear or branched amine or polyalkyleneamine where at least one of the amine hydrogens has been substitutedwith a CR₃ R₄ CO₂ H group; or

a pharmaceutically acceptable salt thereof; with a compound B and analdehyde or aldehyde precurser equivalent, where B represents a linearor branched amine or polyalkylene amine where there is at least oneamine hydrogen;

in the presence of caustic and a suitable solvent, at a temperature of20° C. or less, followed by heating and separating the desired productof formula I, II or III;

B) reacting the product obtained from Step (A) with a halo-(CR₁ R₂)_(n)CR₃ R₄ acid, at a pH of 9 or higher, in the presence of caustic, at atemperature of 20° C. or less, to provide the compounds of formula I, IIor III where at least one of R₁, R₂, R₃ and R₄ is CO₂ H;

C) hydrolyzing the product of Step (B) where Z is NHC(O)CH₃ with NaOH inH₂ O, to provide the products of formula I, II or III where Z is NH₂ ;

D) reacting the product obtained from Step (A) with glyeolonitrile, incaustic, at a pH of 9 or higher, at a temperature of 20° C or less,followed by hydrolysis of the cyano group with HC1 in H₂ O, to providethe products of formula I, II or III where at least one of R₁, R₂, R₃and R₄ is CO₂ H;

E) hydrolyzing the product of Step (A) where Z is NHC(O)CH₃ with DC1 inD₂ O, with heating, to provide the products of formula I, II or IIIwhere Z is NH₂ ; and

F) reacting the product obtained from any one of Steps (A) through (E),where Z is NH₂, with thiophosgene to provide the products of formula I,II or III where Z is isothiocyanato.

The reaction conditions and reagents for the various steps above are asfollows. When the temperature is "20° C. or less" this is usuallyaccomplished by use of an ice/water bath. "Heating" is done either atreflux or above room temperature. The preferred "caustic" is sodiumhydroxide, but any suitable base the is able to maintain the desired pHwithout adverse effect on the product formed from the reaction isacceptable. A "suitable solvent" is inert and provides solubility forthe reactants, examples of such solvents are water, and alcohols such asmethanol. The desired product may be seperated by any conventionalmethods, for example precipitation from a solvent such as acetone.

The complexes of formula I, II or III are prepared by conventionalmethods, for example by reacting the chelant with the metal underconditions such that the metal is sequestered by the chelant.Frequently, the chelant is in excess to that of the metal.

The conjugates of formula I, II or III are prepared by conventionalmethods, for example by covalently attaching the complex to an antibodyor antibody fragment.

The invention will be further clarified by a consideration of thefollowing examples, which are intended to be purely exemplary of the useof the invention. Structures of compounds with reference to the genericformula I are shown in Table I.

Preparation of Starting Materials

EXAMPLE A. PREPARATION OF UNSYMMETRICAL ETHYLENE-DIAMINEDIACETIC ACID.

Deionized water (60.6 g), 98% N-acetylethylenediamine (20.4 g, 0.2mole), and bromoacetic acid (55.7 g, 0.40 mole) were added to a reactionvessel and cooled in an ice-water bath. The pH of the mix was adjusted,while stirring, to approximately 8.1 with 25% sodium hydroxide solution.The temperature of the mix was maintained at less than 20° C. during thecaustic addition. The ice-water bath was removed and the pH maintainedbetween 7 and 8 by the addition of 25% sodium hydroxide solution. Thetemperature was controlled at less than 37° C. by periodically coolingwith an ice-water bath. The reaction mixture was stirred and maintainedas above for approximately 31 hours and then transferred to around-bottom reaction flask equipped with a water-cooled refluxcondenser, magnetic stirrer bar, thermometer, addition funnel and aheating mantle. Sodium hydroxide solution (40.1 g of 50% solution) wasadded and the mix heated, with stirring, at reflux for approximately 15hours and then cooled and filtered using a medium glass frit funnel andvacuum. The filtrate was transferred quantitatively (using deionizedwater) to a beaker and cooled in an ice-bath to less than 25° C.Deionized water (100 ml) was added with stirring and the pH adjusted toapproximately 4 with concentrated hydrochloric acid while maintainingthe temperature at less than 25° C. The mix was filtered using a mediumglass frit funnel and vacuum. Approximately 1200 ml of ethanol wereadded to a large beaker and stirred with a magnetic stirrer bar. Thefiltrate from above was added to the ethanol with thorough mixing. Anoily material forms which gradually turns into a white solid. Agitationwas continued for two hours at which time the solids were collected byfiltering using a medium glass frit funnel and vacuum. The solids wereallowed to dry by exposure to air for about 1.5 hours and then placed ina vacuum oven and dried at 55°-60° C. for several hours. Approximately42.9 g of white solids containing inorganic salt were collected andidentified as unsymmetrical ethylenediaminediacetic acid by proton andcarbon NMR.

EXAMPLE B. PREPARATION OF 2-OXO-1-PIPERAZINEACETIC ACID; LACTAM OFETHYLENEDIAMINEDIACETIC ACID.

Deionized water (150 g), 25.0 g (0.14 mole) of symmetricalethylenediaminediacetic acid, and 28 g of concentrated hydrochloric acidwere added to a round-bottom reaction flask equipped with a thermometer,temperature controller, water-cooled reflux condenser, and heatingmantle. The mixture was stirred with a magnetic stirrer bar and heatedat reflux for four hours and cooled. The contents were filtered using amedium glass frit funnel and vacuum. The pH of the filtrate was adjustedto approximately 1.5 with 50% sodium hydroxide solution and filteredwith a medium glass frit funnel using vacuum. The pH of the filtrate wasadjusted to about 5 with 50% sodium hydroxide solution and the volatilesremoved (in vacuo) at a temperature of 60°-70° C. The solids were driedin a vacuum oven at 55°-60° C. for several hours. The lactam ofsymmetrical ethylenediaminediacetic acid was confirmed by proton andcarbon NMR.

EXAMPLE C. PREPARATION OF 2-OXO-1,4-PIPERAZINEDIACETIC ACID; LACTAM OFETHYLENEDIAMINETRIACETIC ACID.

Approximately 40.8 g of 2-oxo-l-piperazineacetic acid, prepared by theprocedure of Example B, and 70 g of deionized water were added to abeaker and stirred for several hours with a magnetic stirrer bar. Thecontents were filtered using a medium glass frit funnel and vacuum. Thefiltrate and 20.0 g of bromoacetic acid were added to a beaker andstirred till all the bromoacetic acid had dissolved. The pH was adjustedto approximately 7 with 25% sodium hydroxide solution. The temperaturewas maintained at less than 25° C. during the caustic addition bycooling in an ice-water bath. The ice-water bath was removed and the mixallowed to stir for approximately 4-5 hours at approximately 35° C.while maintaining the pH at about 7 by the periodic addition of 25%sodium hydroxide solution. The reaction mix was allowed to stand forseveral hours and then concentrated (in vacuo) to a weight ofapproximately 90-100 g and filtered using a medium glass frit funnel andvacuum. Volatiles were removed (in vacuo) from the filtrate at atemperature of 55°-60° C. and the material dried in a vacuum oven at55°-60° C. for several hours. The lactam of ethylenediaminetriaceticacid was confirmed by proton and carbon NMR.

EXAMPLE D. PREPARATION OF TRISODIUM ETHYLENEDIAMINETRIACETIC ACID.

Approximately 44.5 g of the crude 2-oxo-l,4-piperazinediacetic acid,prepared by the procedure of Example C, and 280 g of deionized waterwere added to a beaker and agitated till the lactam had dissolved.Caustic solution (110 g, 50%) was added with agitation. The temperaturewas maintained at less than 25° C. by cooling in an ice-bath. Hydrolysiswas then achieved by immersing tubes containing the solution into awater bath controlled at 87° C. After 15 minutes, the solutions wereremoved and cooled in an ice-water bath. Analysis by proton and carbonNMR confirmed the presence of trisodium ethylenediaminetriacetic acid inthe alkaline hydrolysis medium.

EXAMPLE E. PREPARATION OF 4-DIETHYLENETRIAMINEACETIC ACID.

To a flask equipped with a water-cooled reflux condenser, magneticstirrer and thermometer were added 75.0 g of phthalic anhydride, 350.5 gof acetic acid and 26.0 g of diethylenetriamine. The mix was stirred andheated at approximately 116° C. for 1.5 hours and then cooled. Volatileswere removed under vacuum at 65°-70° C. until a weight of 218 g wasobtained. The mixture was poured into 600 g of ethanol with stirring.After two hours the solids were filtered using a medium glass fritfunnel. The solids were washed twice with 500 ml of ethanol and thendried in a vacuum oven at 60°-65° C. Approximately 66 g of material ofthe diphthaloyl compound were collected.

The ethyl ester of the diphthaloyl compound was prepared by adding 65.6g of the above prepared diphthaloyl compound, 17.7 g of sodium carbonateand 800 ml of ethanol to a flask equipped with a water-cooled refluxcondenser, addition funnel, mechanical stirrer and a thermometer with atemperature controller. Ethyl bromoacetate (51.0 g) was added over a 15minute period to the stirred mixture and then heated at reflux for 16hours. Ethanol was removed (200 ml) by distillation using a Dean-Starkdistillation trap and the remaining reaction mixture cooled to less than5° C. by the addition of crushed ice. The mixture was cooled for anadditional 5 hours in an ice bath and filtered using a medium glass fritfunnel. The solids were washed twice with ethanol and dried in a vacuumoven at 65°-70° C. Approximately 81 g of ethyl1,7-diphthaloyl-4-diethylenetriamineacetate were obtained. In 0.32 g ofwater and 76.4 g of concentrated hydrochloric acid was dissolved 20.1 g(0.045 moles) of ethyl 1,7-diphthaloyl-4-diethylenetriamineacetate withheating to 93° C. and the mixture held at 93° C. for 6.5 hours. Theresulting white precipitate was filtered and washed with water. Thecombined filtrate was concentrated at 60° C. under vacuum to give awhite solid. NMR analysis indicated that the phthaloyl groups were notcompletely hydrolyzed. The two solids were then combined and added toconcentrated hydrochloric acid with a small amount of water. The slurrywas then heated to reflux for 6 hours, cooled to room temperature andfiltered to give 12.3 g of phthalic acid. The filtrate was thenevaporated under vacuum to give 13.9 g of product as a yellow solid. Theproduct was dissolved in water by the addition of 6 g of 50% sodiumhydroxide and treated with activated charcoal at 100° C. followed byfiltration and evaporation under vacuum to give 15.2 g4-diethylenetriamineacetic acid.

Preparation of Final Products EXAMPLE 1. PREPARATION OF2-[(2-{[BIS(CARBOXYMETHYL)]AMINO}ETHYL ) AMINO]-2-(5-ACETAMIDO-2-HYDROXYPHENYL )-ETHANOI C ACID.

Deionized water (10.3 g), 98% 4-acetamidophenol (15.1 g 0.1 mole), 50%aqueous glyoxylic acid (14.8 g, 0.1 mole), and methanol (50.5 g) wereadded to a beaker and mixed using a magnetic stirrer bar. Unsymmetricalethylenediaminediacetic acid (19.5 g), prepared by the procedure ofExample A, was added and the mix cooled in an ice-water bath. The pH ofthe mix was adjusted, while stirring, to approximately 8.0 with 50%sodium hydroxide solution. The temperature of the mix was maintained atless than 20° C. during the caustic addition. The ice-water bath wasremoved and the mix adjusted to pH 8.7 and stirred at 25°-32° C. forapproximately 2 hours. The mix was transferred to a round-bottomreaction flask equipped with a water-cooled reflux condenser, magneticstirrer bar, thermometer, and a heating mantle. The mix was heated, withstirring, at 70° C. for 8 hours and then cooled and filtered using amedium glass frit funnel and vacuum. The solids were allowed to dry byexposure to air for 7 hours and then placed in a vacuum oven and driedat 55°-60° C. for several hours. Approximately 29.6 g of solids werecollected. The material was then agitated with approximately 300 g ofacetone and filtered using a medium glass frit funnel and house vacuum.The solids were washed once again with an additional 300 g of acetone,air dried and then placed in a vacuum oven for one hour at 55°-60° C.Approximately 26.7 g of2-[(2-{[bis(carboxymethyl)]-amino}ethyl)amino]-2-(5-acetamido-2-hydroxyphenyl)ethanoicacid, sodium salt were collected. These solids and 180 g of deionizedwater were placed in a beaker and stirred with a magnetic stirrer bar.The pH was adjusted to 2.2 with concentrated hydrochloric acid at whichpoint the acid form of the product began to precipitate from solution.The product was collected by filtration and washed with approximately150 g of deionized water. The product,2-[(2-{[bis(carboxymethyl)]amino}ethyl)amino]-2-(5-acetamido-2-hydroxyphenyl)ethanoicacid was dried in a vacuum oven at 55°-60° C. for several hours.Approximately 14.2 g of product were obtained. Proton NMR verified thestructure of the product. (See Table I.)

EXAMPLE 2. PREPARATION OF 2-[(2-{[BIS(CARBOXYMETHYL)]-AMINO} ETHYL)(CARBOXYMETHYL)AMINO]-2-[5-ACETAMIDO-2-(CARBOXYMETHYLOXY)PHENYL]ETHANOICACID.

Deionized water (4.5 g), bromoacetic acid (2.0 g) and2-[(2-{[bis(carboxymethyl)]amino}ethyl)amino]-2-(5-acetamido-2-hydroxyphenyl)ethanoic acid (2.5 g), prepared by the procedure of Example 1, wereadded to a small reaction vessel and cooled in an ice-water bath. The pHof the mix was adjusted, while stirring, to approximately 9.3 with 25%sodium hydroxide solution. The temperature of the mix was maintained atless than 20° C. during the caustic addition. The ice-water bath wasremoved and the mix allowed to stir for 48 hours at a temperature of35°-40° C. while maintaining the pH between 10.5 and 11.5 by theperiodic addition of 25% sodium hydroxide solution. A portion of thereaction mixture (10.2 g) was added to a beaker and stirred with amagnetic stirrer bar. Acetone (125 g) was added to the solution over a15-minute period resulting in the precipitation of an oil. The acetoneportion was removed by decanting and an additional 50 g of acetone addedto the precipitate, mixed, and the acetone layer removed. The oil wasair dried and then dried in a vacuum oven at 60°-65 ° C. for about twohours to give a crispy yellow solid. The product was purified by anionexchange chromatography on Q-Sepharose™ from Pharmacia Inc. on a 15 mm×500 mm column eluting with a gradient of 0-30% formic acid over twohours at a rate of 3 ml/min and collecting fractions. The fractions weremonitored by UV absorption and the appropriate fractions combined andlyophilized to give the desired product. (See Table I.)

EXAMPLE 3. PREPARATION OF 2-[(2-{[BIS(CARBOXYMETHYL)]AMINO}ETHYL)(CARBOXYMETHYL)AMINO]-2-[5-AMINO-2-(CARBOXYMETHYLOXY)PHENYL]ETHANOICACID, PENTA SODIUM SALT.

Approximately 40 mg of 2-[(2-{[bis(carboxymethyl)]amino}ethyl)(carboxymethyl)amino]-2-[5-acetamido-2-(carboxymethyloxy)phenyl]ethanoicacid, prepared by the procedure of Example 2, was dissolved in 700 μl ofD₂ O and adjusted with NaOD/D₂ 0 to pH 13. Hydrolysis of the N-acetylgroup to the corresponding aniline functionality proceeded at ambienttemperature and was followed by proton NMR which confirmed thestructure. (See Table I.)

EXAMPLE 4. PREPARATION OF 2-[(2-{IBIS(CARBOXYMETHYL)]AMINO}ETHYL)(CYANOMETHYL)AMINO]-2-(5-ACETAMIDO-2-HYDROXYPHENYL)ETHANOIC ACID.

Deionized water (3.1 g) and 2.5 g of 2-8(2{[bis(carboxymethyl)]amino}ethyl)amino]-2-(5-acetamido-2-hydroxyphenyl)ethanoic acid, prepared by theprocedure of Example 1, were added to a small glass vessel and cooled inan ice-water bath. The pH was adjusted to 9.8-9.9 with 25% sodiumhydroxide solution. The temperature of the mix was maintained at lessthan 20° C. during the caustic addition. The ice-bath was removed and1.0 g of an aqueous 40% glycolonitrile solution was added with mixingand the pH adjusted to 9.9-10.0 with 25% sodium hydroxide solution. Themix was transferred to a small reaction flask equipped with athermometer containing a temperature controller, water cooled refluxcondenser, and heating mantle. The reaction mixture was stirred with amagnetic stirrer bar and heated at 49°-50° C. for eight hours, cooledand allowed to stand at ambient temperature for 72 hours. A portion ofthe reaction mixture (8.5 g) was added to a beaker and stirred with amagnetic stirrer bar. Acetone (146 g) was added to the solution over a10-minute period, resultng in the precipitation of a solid material. Theacetone portion was removed by decanting and an additional 50 g ofacetone added to the precipitate, mixed, and the acetone layer removed.The material was dried in a vacuum oven at 60°-65° C. for about fourhours. Approximately 2.9 g of product was collected. Proton NMRsupported the desired aminoacetonitrile derivative. (See Table I.)

EXAMPLE 5. PREPARATION OF 2-[(2-{[BIS(CARBOXYMETHYL)]-AMINO}ETHYL)(CARBOXYMETHYL)AMINO]-2-(5-AMINO-2HYDROXYPHENYL)ETHANOIC ACID.

Approximately 1.0 g of 2-[(2-{[bis(carboxymethyl)]amino}ethyl)(cyanomethyl)amino]-2-(5-acetamido-2-hydroxyphenyl)ethanoic acid,prepared above by the procedure of Example 4, was hydrolyzed underacidic conditions to convert the aminoacetonitrile functionality to thecorresponding acetate group and the N-acetyl group to the aniline group.The aminoacetonitrile compound, 2.2 g of D₂ O, and 7.8 g of 20% DC1 wereadded to a glass tube. The tube was placed in a temperature-controlledwater bath at 88°-89° C. for a total of 33 minutes and then removed andcooled. The hydrolysis was followed by proton NMR. The solution was thenfreeze-dried and lyophilized to give 1.3 g of solids. The product waspurified by anion exchange (Q-Sepharose™) on a 15 mm ×500 mm columneluting with a gradient of 0-1M acetic acid over one hour at a rate of 3ml/min and collecting 6 ml fractions. The fractions were monitored by UVabsorption and the appropriate fractions were combined and lyophilizedto give the desired product. (See Table I.)

EXAMPLE 6. PREPARATION OF 2-[BIS(2-{[(BIS(CARBOXYMETHYL)]AMINO}ETHYL)AMINO]-2-[5-ACETAMIDO-2-(CARBOXYMETHYLOXY)PHENYL]ETHANOIC ACID AND2-[{2-[(2-{[BIS(CARBOXYMETHYL)]AMINO}ETHYL) (CARBOXYMETHYL)AMINO]ETHYL}(CARBOXYMETHYL)AMINO]-2-[5-ACETAMIDO-2-(CARBOXYMETHYLOXY)PHENYL]ETHANOICACID.

Deionized water (24.8 g), 15.1 g (0.1 mole) of 98% 4-acetamidophenol,and 14.8 g of 50% aqueous glyoxylic acid (0.1 mole) were added to abeaker and cooled in an ice-water bath. The pH of the mix was adjustedto 3.3 with 25% sodium hydroxide solution while maintianing thetemperature at less than 20° C. DETA (9.8 g) was then added. Once againthe temperature was kept below 20° C. by cooing in an ice-water bath.The pH after addition of the DETA was approximately 10.2. The mix wastransferred to a reaction flask equipped with a thermometer, atemperature controller, water-cooled reflux condenser, and heatingmantle. The reaction mixture was stirred with a magnetic stirrer bar andheated at 75° C. for approximately seven hours and cooled. Acetone (1400g) was added to a large beaker and stirred with a magnetic stirrer bar.Approximately 40 g of the reaction solution prepared above was addedover a 10 minute period resulting in the precipitation of a solidmaterial. The acetone portion was removed by decanting and an additional1460 g of acetone added and the solid titurated and mixed thoroughlyunder acetone. The solids were recovered by filtering using a mediumglass frit funnel and vacuum. The solids were washed with a copiousamount of acetone and then dried in a vacuum oven at a temperature of60°-65° C. for several hours. Approximately 7.8 g of solids wererecovered with proton NMR showng a mixture of the desired isomers of theDETA compound.

Deionized water (5.3 g) and 4 g of the above isolated solids were addedto a beaker and stirred with a magnetic stirrer bar for about threehours at which time the solids had completely dissolved. Bromoaceticacid (10.1 g) was added with stirring and the mix cooled in an ice-waterbath. The pH of the mix was adjusted to approximately 11 with 25% sodiumhydroxide solution. The temperature was maintained at less than 20° C.during the caustic addition. The ice-water bath was removed and the mixallowed to stir for 50 hours at a temperature of 35°-40° C. whilemaintaining the pH between approximately 10.5-11.5 by the periodicaddition of 25% sodium hydroxide solution. Acetone (240 g) was added toa beaker and stirred with a magnetic stirrer bar. Approximately 5 g ofthe reaction solution was added to the acetone resulting in theprecipitation of a solid. The acetone portion was decanted and anadditional 245 g of acetone added, mixed and the acetone layer removed.The solids were collected by filtering using a medium glass frit funneland vacuum. The solids were washed with acetone and then dried in avacuum oven at 55°-60° C. for several hours. Approximately 2.6 g ofsolids were collected. (See Table I.)

EXAMPLE 7. PREPARATION OF 2-[BIS(2-{[(BIS(CARBOXYMETHYL)]AMINO}ETHYL)AMINO]-2-[5-AMINO-2-(CARBOXYMETHYLOXY)PHENYL]ETHANOIC ACID AND2-[{2-[(2-{[BIS(CARBOXYMETHYL)]AMINO}ETHYL)(CARBOXYMETHYL)AMINO]ETHYL}(CARBOXYMETHYL)AMINO]-2-[5-AMINO-2-(CARBOXYMETHYLOXY)PHENYL]ETHANOICACID.

Approximately 376 mg of 2-[bis(2-{[(bis(carboxymethyl)]amino}ethyl)amino]-2-[5-acetamido-2-(carboxymethyloxy)phenyl]ethanoic acid and2-[{2-[(2-{[bis(carboxymethyl)]amino}ethyl)(carboxymethyl)amino]ethyl}(carboxymethyl)amino]-2-[5-acetamido-2-(carboxymethyloxy)phenyl]ethanoicacid, prepared by the procedure of Example 6, was dissolved in 1.0 g ofD20 and treated with 5 drops of 37% DC1. The acidic solution was thenheated at 80° C. for 2 hours after which time the proton NMR spectrumindicated that virtually all of the acetanilide groups had beenconverted to aniline groups and acetic acid. The solution was thenfrozen in a dry-ice acetone bath and lyophilized overnight to yield thedesired product as a light brown solid. (See Table I.)

EXAMPLE 8. PREPARATION OF 2-[{2-[(2-{[BIS(CARBOXYMETHYL)]-AMINO}ETHYL)(CARBOXYMETHYL) AMINO]ETHYL}(CARBOXYMETHYL)AMINO ]-2-[5-AMINO-2-CARBOXYMETHYLOXY)PHENYL]ETHANOIC ACID.

In 40 ml of water was dissolved 8.0 g of 4-diethylenetriamineaceticacid, prepared by the procedure of Example E, and then the mixture wascooled in an ice bath. To this cooled solution was added 6.08 g (0.04moles) of 4-acetamidophenol and a chilled solution of 5.95 g (0.04moles) of a 50 weight % solution of glyoxylic acid in water. Whilekeeping the slurry at less than 20° C. with the ice bath, a 2.5 mlportion of 50 weight % sodium hydroxide was added. The resulting slurryat pH 8.75 was heated slowly to 80° C., held at this temperature for 4.5hours with stirring, then allowed to cool overnight. The solution wasthen evaporated under vacuum to about 25 ml volume and added to 300 mlof acetone. The acetone was decanted from the resulting solid. The solidwas washed several times with acetone and dried to give 26.1 g ofproduct as a dark sticky solid. A 26.05 g portion of this solid wasdissolved in 50 ml of water. Into this solution was dissolved 26.7 g(0.192 moles) of bromoacetic acid. The resulting solution was cooled inan ice bath, the pH adjusted to 10.5 with 50 weight % sodium hydroxide,allowed to warm to room temperature, and then heated to 46° C. Thetemperature was kept at 46° C. and the pH kept at 10.5 by addition of 50weight % sodium hydroxide for about 23 hours. The volume was thenreduced to 50 ml under vacuum. The concentrated solution was added to500 ml of acetone with vigerous stirring and the resulting precipitateallowed to settle. The acetone was decanted and an additional 400 ml ofacetone was added, vigerously stirred and then decanted. A final wash inthe same manner using 100 ml of acetone was done. The solid was driedunder vacuum to give 52.55 g of crispy brown solid. A 2.00 g sample ofthis brown solid was dissolved in 20 ml of water and treated with 1.48 gof concentrated hydrochloric acid. This solution was heated at 80° C.until analysis by proton NMR indicated complete hydrolysis of theN-acetyl moiety. The solution was then freeze dried to give 2.13 g ofbrown solid containing the title product. (See Table I.)

EXAMPLE 9. PREPARATION OF2-[(2-[(2-[(2-{[BIS(CARBOXYMETHYL)]AMINO}ETHYL)(CARBOXYMETHYL)AMINO]ETHYL)(CARBOXYMETHYL)AMINO]ETHYL)(CARBOXYMETHYL)AMINO]-2-[5-ACETAMIDO2-(CARBOXYMETHYLOXY)PHENYL]ETHANOIC ACID AND2[(2-[(2-{[BIS(CARBOXYMETHYL)]AMINO}ETHYL)(CARBOXYMETHYL)AMINO]ETHYL)(2-{[BIS(CARBOXYMETHYL)]AMINO}ETHYL)AMINO]-2-[5-ACETAMIDO-2-(CARBOXYMETHYLOXY)PHENYL]ETHANOIC ACID.

Deionized water (12.5 g), 98% 4-acetamidophenol (7.6 g), and 50% aqueousglyoxylic acid solution (7.4 g) were added to a beaker and cooled in anice-water bath. The pH of the mix was adjusted to 3.6 with 25% sodiumhydroxide solution while maintaining the temperature at less than 20° C.Linear triethylenetetraamine (7.2 g) was added while keeping thetemperature below 20° C. The pH after addition of thetriethylenetetraamine was approximately 10.6. The mix was transferred toa reaction flask equipped with a thermometer containing a temperaturecontroller, water cooled reflux condenser, and heating mantle. Thereaction mixture was stirred with a magnetic stirrer bar and heated at80°-83° C. for 4.5 hours and cooled. Acetone (175 g) was added to abeaker and stirred with a magnetic stirrer bar. Approximately 12 g ofthe reaction solution was added resulting in the precipitation of anoil. The acetone portion was removed by decanting and an additional 175g of acetone addded and stirring continued. The acetone portion wasremoved and the precipitant dried in a vacuum oven at 60°-65° C. forseveral hours. Approximately 3.1 g of solids were collected. The solidswere then slurried in 100 g of acetone, thoroughly mixed and filteredusing a medium glass frit funnel and vacuum. The solids were then washedwith an additional 250 ml of acetone and dried once again in a vacuumoven at 60°-65° C. for about four hours. Approximately 2.0 g of materialwas recovered with proton NMR indicating a mixture of thetriethylenetetraamine isomers present.

Deionized water (2.0 g) and 1.86 g of the above solid product were addedto a beaker and stirred for one hour at which time the solids weremostly dissolved. Bromoacetic acid (5.0 g) was added with stirring andthe mix cooled in an ice-water bath. The pH of the mix was adjusted toapproximately 10.5 and maintained for 47 hours at a temperature of35°-40° C. while maintianing the pH between 10.5-11.5 by the periodicaddition of 25% sodium hydroxide solution. Acetone (130 g) was added toa beaker and stirred with a magnetic stirrer bar. Approximately 10.8 gof the reaction solution was added to the acetone resulting in theprecipitation of a solid. The acetone portion was decanted and anadditional 150 g of acetone added to the precipitate, mixed, and theacetone layer removed. The solids were dried in a vacuum oven at 60°-65°C. for several hours. Approximately 7.2 g of solids were collected. (SeeTable I.)

EXAMPLE 10. PREPARATION OF2,6-BIS{[BIS(CARBOXYMETHYL)AMINO](CARBOXY)METHYL}-4-(ACETAMIDO) PHENOL

To a beaker was added 38.6 g of 98% 4-acetamidophenol, 35.3 g of 98%iminodiacetic acid, 150 mls. of methanol, 38.5 g of 50% aqueousglyoxylic acid solution, and 30 g of deionized water. The mix was cooledin an ice-water bath and the pH adjusted while mixing to approximately9.4 with 50% sodium hydroxide solution. The temperature was maintainedat less than 30° C. during the caustic addition. The mix was transferredto a reaction flask equipped with a water cooled reflux condenser,thermometer, and heating mantle. The reaction mixture was heated toapproximately 74°-76° C. and the pH monitored and kept between 8.7-9.5by the periodic addition of 50% sodium hydroxide solution. The mix washeated for a total of 18 hours. During this time approximately 40 g ofdeionized water were added. After cooling, the reaction mix was filteredusing a medium glass frit and vacuum. Deionized water (75 g) was addedto the filtrate and the methanol removed in vacuo at ambient temperature(about 20°-25° C.). The solution was allowed to stand for several hoursand the precipitated solids removed from solution by filtering using amedium glass frit funnel and vacuum. Approximately 30 g of the filtrateand 15 g of ethyl ether were mixed thoroughly and the ether layer thenseparated. The process was repeated using 15 g and 10 g of ethyl etherin succession. The aqueous layer was adjusted with aqueous hydrochloricacid solution to a pH of about 0.5 and volatiles removed in vacuo at atemperature of 50°-55° C. Approximately 13.5 g of solids were collected.Methanol (75 g) was added to the solids and the insoluble salts removedby filtration. Methanol was removed (in vacuo) and the remaining solidsdried in a vacuum oven at 70°-75° C. for several hours. The product,which still contained some inorganic salt, was analyzed by proton NMRand found to be predominately the bis-substituted product. (See TableI.)

EXAMPLE 11. PREPARATION OF 2,6-BIS{[(2{[BIS(CARBOXYMETHYL)]AMINO}ETHYL)(CARBOXYMETHYL)]AMINOMETHYL}-4-(ACETAMIDO) PHENOL.

The alkaline trisodium ethylenediaminetriacetic acid solution, preparedby the procedure of Example D, was cooled in an ice-bath andhydrochloric acid added with stirring to obtain a pH of about 13.8. Thetemperature was maintained at less than 35° C. during the acid addition.Volatiles were removed (in vacuo) at ambient temperature to a weight of210 g. The solids were removed by filtering on a medium glass fritfunnel using vacuum. The filtrate was transferred to a 250 mlround-bottom flask equipped with a water-cooled reflux condenser,magnetic stirrer bar, thermometer, temperature controller, heatingmantle, and addition funnel. The pH was adjusted to about 11 withhydrochloric acid. The temperature was maintained at less than 30° C.during the acid addition. The mix was heated to approximately 40° C. and11.6 g of 37% aqueous formaldehyde solution added dropwise from theaddition funnel over a 35 minute period. The reaction mixture wasstirred and heated for an additional 30 minutes and then cooled. Thesolution was adjusted with 25% sodium hydroxide solution to a pH ofabout 9.8 and transferred to an addition funnel. To a beaker was added10.3 g of 98% 4acetamidophenol, 25.2 g of deionized water, and 9.5 g of25% sodium hydroxide solution. The mix was stirred till completedissolution was obtained. The solution was transferred to a round-bottomreaction flask equipped as described above and heating and stirringinitiated. The mix was heated to approximately 65° C. at which point theformaldehyde adduct solution prepared above was added dropwise overapproximately a one hour period. The reaction was stirred and heated at65° C. for an additional 12 hours and then cooled. Acetone (150 g) wasadded to a beaker and stirred with a magnetic stirrer bar. Approximately10 g of the crude reaction mixture was added to the acetone resulting inthe precipitation of an oily material. The acetone portion was decantedand an additional 150 g of acetone added to the precipitate, mixed, andthe acetone layer removed. The material was dried in a vacuum oven at55°-60° C. for several hours. Approximately 3.1 g of solids werecollected. Approximately 165 mg of the solids were dissolved in aminimum of water and loaded onto a Q-Sepharose™ (from Pharmacia Inc.)column [1.5 cm × 50 cm., acetate form]and eluted using a gradient of 0to 1M ammonium acetate over two hours at 2 ml/min. The absorbance at 300nm was observed. The product was contained in the third major peak. Thiswas isolated and freeze-dried to yield 36.4 mg of solids which wascharacterized by proton and carbon NMR and fast atom bombardment massspectrometry as 2,6bis{[(2-{[bis(carboxymethyl)]amino}ethyl)(carboxymethyl)]aminomethyl}-4-(acetamido)phenol.(See Table I.)

EXAMPLE 12. PREPARATION OF 2,6-BIS{[(2{IBIS(CARBOXYMETHYL) ]AMINO}ETHYL)(CARBOXYMETHYL) ]AMINOMETHYL}-4-(AMINO) PHENOL.

Approximately 264 mg. of2,6-bis{[(2-{[bis(carboxymethyl)]amino}ethyl)(carboxymethyl)]aminomethyl}-4(acetamido)phenol,prepared by the procedure of Example 11, was placed in a 5 mm NMR tubeand dissolved in a mixture of D₂ O (0.5 ml) and DCL (0.5 ml, 20%). TheNMR tube was placed in a hot water bath (85° C.) for short periods oftime and the reaction progress monitored by NMR (disappearance of theacetamide methyl protons and appearance of acetic acid). After 35minutes the reaction was complete. The reaction mixture was freeze-driedto yield the crude amine hydrochloride as a dark solid material. Thecrude product was dissolved in a minimum amount of water and loaded ontoa Q-Sepharose^(TM) column (1.5 cm × 50 cm, acetate form) and elutedusing a gradient of 0 to 1M ammonium acetate over three hours at 2ml/min. The absorbance at 300 nm was observed. The product was containedin the third major peak. This was isolated and freeze-dried to leave apale amber solid (122 mg) which was a mixture of the desired amineproduct and ammonium chloride. The product mixture was characterized byproton and carbon NMR and elemental analysis. The salt-containingproduct (250 mg from combined batches) was further purified onQ-Sepharose^(TM) (1.5 cm × 50 cm, formate form) using a gradient of 0 to10% formic acid over four hours. The absorbance at 300 nm was observed.The first major peak contained the desired product. This was isolatedand freeze-dried to yield 8.3 mg of a white crystalline solid. Thestructure was confirmed by proton and carbon NMR and fast atombombardment mass spectrometry. (See Table I.)

EXAMPLE 13. PREPARATION OF 2,6-BIS{[(2-{[BIS(CARBOXYMETHYL)]AMINO}ETHYL)(CARBOXYMETHYL) ]AMINOMETHYL}-4-(ISOTHIOCYANATO)PHENOL.

Product containing 2,6-bis{[(2-{[bis(carboxymethyl)]amino}ethyl)(carboxymethyl)]aminomethyl}-4(amino)phenol and inorganic salt (208 mg,15% in NH₄ C1), prepared by the procedure of Example 12, was dissolvedin a minimum amount of water and passed through a Sephadex™ G-10(Pharmacia, Inc.) desalting column (1 cm × 35 cm). The salt-free aminewas eluted with water and freeze-dried (11.5 mg). The amine wasdissolved in water (10 ml) and placed in a round-bottom reaction flask.Thiophosgene (0,015 ml, 10 eq) dissolved in methylene chloride (1 ml)was added. The reaction mixture was stirred at room temperature for onehour. The mixture was then washed with several portions of methylenechloride to remove excess thiophosgene and the aqueous layerfreeze-dried to give the crude isothiocyanato product which wascharacterized by fast atom bombardment mass spectrometry. (See Table I.)

EXAMPLE 14. PREPARATION OF2-({[BIS(CARBOXYMETHYL)]AMINO}METHYL)-4-(ACETAMIDO) PHENOL.

Deionized water (35.3 g), 35.3 g of 98% iminodiacetic acid (0.25 mole),and 29.9 g of 50% aqueous sodium hydroxide solution were weighed into around-bottom reaction flask equipped with a water-cooled refluxcondenser, mechanical stirrer, thermometer with a temperaturecontroller, and an addition funnel. The mix was heated, with stirring,to a temperature of 55° C. Aqueous 37% formaldehyde solution (21.5 g)was placed in the addition funnel and added to the reaction flask over a15 minute period. The reaction mixture was heated at 55° C. forapproximately 45 minutes, cooled and transferred to an addition funnel.To a round-bottom flask equipped as above was added 38.7 g (0.25 mole)of 98% 4-acetamidophenol, 35.3 g of deionized water, and 12.2 g of 50%aqueous sodium hydroxide solution. The mix was heated, with stirring, toa temperature of approximately 65° C., and theformaldehyde-iminodiacetic acid adduct solution added over a 30 minuteperiod. The reaction mixture was heated at 65° C. for an additionaltwelve hours and cooled. Concentrated hydrochloric acid (55.5 g) wasadded and the reaction mixture stirred for one hour. The solution wasallowed to stand for several weeks and then the crystalline precipitatefiltered, washed with deionized water and dried in a vacuum oven at 65°C. for several hours. Approximately 17.4 g. of solids were recovered.The structure was confirmed by proton NMR. (See Table I.)

EXAMPLE 15. PREPARATION OF2-({[BIS(CARBOXYMETHYL)]AMINO}METHYL)-6-{[({[BIS(CARBOXYMETHYL)]AMINO}ETHYL)((CARBOXYMETHYL)AMINO]METHYL}-4-(ACETAMIDO)PHENOL.

Approximately 5.7 g of crude 2-oxo-l,4-piperazinediacetic acid, preparedby the procedure of Example C, and 38.6 g of deionized water were addedto a beaker and mixed till dissolution of the lactam was achieved.Caustic solution (13.5 g of 50% solution of sodium hydroxide) was addedwhile maintaining the temperature at less than 30° C. by cooling in anice water bath. The solution was then transferred to a glass tube andimmersed in a 90° C. water bath for 10 minutes and then cooled in an icewater bath. The conversion of the lactam to the trisodium salt ofethylenediaminetriacetic acid was confirmed by proton NMR. The alkalinesolution was then adjusted to a pH of approximately 11.9 by the additionof hydrochloric acid. The temperature was maintained at less than 25° C.by cooling in an ice water bath. The solution was transferred to areaction vessel and 1.5 g of aqueous 37% formaldehyde solution addeddropwise over a 20 minute period. A small amount of aqueous causticsolution was also added during this time for pH adjustment. The mix wasstirred for an additional hour with periodic additions of aqueous sodiumhydroxide to maintain the pH between 1.0-11.5.

To a separate reaction vessel was added 1.5 g of 2- ({1b is(carboxy-methyl)]amino}methyl)-4-(acetamido)phenol prepared by theprocedure of Example 12, and 2.5 g of deionized water. Aqueous 25%sodium hydroxide was added, while cooling in an ice bath, to obtain a pHof about 11. The formaldehyde adduct solution prepared above was thenadded over a 30 minute period to the phenolic compound at a temperatureof approximately 30° C. The reaction mixture was mixed and heated for anadditional 10 hours at 70° C. and then cooled. Acetone (100 g) was addedto a beaker and stirred with a magnetic stirrer bar. Approximately 10 gof the crude reaction mixture was added to the acetone resulting in theprecipitation of a gummy material. The acetone portion was decanted andan additional 50 g of acetone added to the material and the producttiturated under acetone. The acetone layer was removed by decanting andthe solids dried in a vacuum oven at 60°-65° C. for several hours. Thedesired product was isolated from the crude mixture by passing anaqueous solution of the solids over a Q-Sepharose™ column and isolatingthe desired fraction as in Example 11. (See Table I.)

EXAMPLE U. PREPARATION OF N,N'-DI(2-HYDROXY,5-ACETAMIDOBENZYL)ETHYLENEDIAMINE-N,N'-DIACETIC ACID. (Comparative)

Ethylenediamine-N,N'-diacetic acid (10 g, 0.056 mole), 25 g of deionizedwater, 7.0 g of 50% sodium hydroxide solution, and 5.0 g of methanolwere added to a round-bottom reaction flask equipped with a water-cooledreflux condenser, mechanical stirrer, thermometer with a temperaturecontroller, and an addition funnel. The reaction mixture was heated to55° C. Aqueous 37% formaldehyde solution (9.2 g, 0.11 mole) was weighedinto the addition funnel and added over a twenty minute period. Thereaction mixture was heated at 55° C. for one hour and then cooled andtransferred to another addition funnel. To a reaction flask, equipped asabove, were added 17.2 g of 4-acetamidophenol (0.11 mole), 36 g ofdeionized water, 2.0 g of 50% sodium hydroxide solution, and 36 g ofmethanol. The mix wax heated to 65° C. and the aqueousformaldehyde/ethylenediamine-N,N'-diacetic acid adduct solution addedover a one hour and fifteen minute period. The reaction mixture washeated an additional 12 hours at 64°-65° C. and then cooled. A portionof the reaction product was concentrated and the methanol removed undervacuum. The solution was adjusted to pH 1.5-2.0 with hydrochloric acidresulting in the precipitation of the acetyl product. The material wasfiltered, washed with deionized water, and dried in a vacuum oven at55°-60° C. for several hours. The structure was confirmed by proton NMR.

EXAMPLE V. PREPARATION OFN,N'-DI(2-HYDROXY-5-AMINOBENZYL)ETHYLENEDIAMINE-N,N'-DIACETIC ACID,HYDROCHLORIDE. (Comparative)

To approximately 0.9 g of the product isolated in Example U were added12.5 g of deionized water and 8 g of concentrated hydrochloric acid. Thesolution was heated at reflux and stirred for one hour in a round-bottomreaction flask. The volatiles were removed (in vacuo), and the aminehydrochloride product dried in a vacuum oven at 50°-60° C. for severalhours. The structure was confirmed by proton NMR.

EXAMPLE W. PREPARATION OFETHYLENEDIAMINEDI(2-HYDROXY-5ACETAMIDOPHENYL)ACETIC ACID]. (Comparative)

Aqueous (50%) glyoxylic acid (30.0 g, 0.20 mole), 98% 4-acetamidophenol(30.9 g, 0.20 mole) and deionized water (22 g) were added to around-bottom reaction flask equipped with a water-cooled refluxcondenser, mechanical stirrer and a thermometer with a temperaturecontroller. The flask was cooled with an ice-water bath and 19.0 g of50% sodium hydroxide solution added slowly with stirring whilemaintaining the temperature below 30° C. Ethylenediamine (6.1 g, 0.10mole) was added at a temperature less than 30° C. The ice bath wasremoved and the reaction mixture heated and stirred for five hours at85°-86° C. Approximately 20 g of the aqueous reaction product wastreated with 10 g of ethyl ether. The ether layer was removed and theprocedure repeated again. The aqueous portion was then adjusted to a pHof approximately 4.2 with hydrochloric acid and agitated with 35 g ofacetone. The acetone layer was removed and discarded. To the remainingmaterial was added 65 g of methanol with stirring. The resulting solidswere filtered and dried in a vacuum oven at 55°-60° C. for severalhours.

EXAMPLE X. PREPARATION OFETHYLENEDIAMINEDI[(2-HYDROXY-5AMINOPHENYL)ACETIC ACID] (Comparative)

To approximately 4.5 g of the above solids were added 6 g of deionizedwater and 21 g of concentrated hydrochloric acid. The mix was filtredand 6 g of water added. The solution was placed in a round-bottomreaction flask equipped with a water-cooled reflux condenser, mechanicalstirrer, and a thermometer. The solution was heated for one hour at100°-103° C. and then cooled. The volatiles were removed in vacuo andthe product, the hydrochloride ofethylenediaminedi(2-hydroxy-5aminophenyl)acetic acid, was dried in avacuum oven at 60° C. for several hours. Hydrolysis of the acetylfunctionality was followed by proton NMR.

COMPLEX PREPARATION AND PERCENT COMPLEX DETERMINATION.

In the following examples the following terms were used: conc. meansconcentrated; OAc means the acetate moiety, OCOCH₃ ; TLC means thinlayer PG,49 chromotography; ambient temperature means room temperatureor about 20° to 25° C.; overnight means from about 9 to 18 hours;SP-Sephadex™ C-25 resin is a cation exchange resin having sulfonic acidfunctionality, sold by Pharmacia, Inc.

The yttrium and/or samarium complexes of several of the compounds wereprepared and percent complexation determined as follows:

Yttrium Complex Preparation:

Complexes were made by preparing a 0.0003M yttrium solution in water.(YCl₃ ·6H₂ O, 303.26 g/mole; Y(OAc)₃, 12.1% H₂ 0). Radioactive YCl₃(Oakridge National Laboratories) was added to give the desired number ofcounts. Ten μl of ligand solution (at 0.03M) were added to 990 μl of theY solution, giving a ligand-to-metal ratio of 1:1. (Ten times the amountof ligand solution was used for a 10:1 ligand to metal ratio.) The pHwas then adjusted to 7.4 using microliter quantities of hydrochloricacid or sodium hydroxide. The solution was then tested for the amount ofcomplexed yttrium using the cation exchange method given below.

Percent Complex Determination:

A disposable 10 ml plastic (Biorad) column was filled with 1 to 2 ml ofwater-swelled Sephadex™ C-25 cation exchange resin. The water waspressure eluted to the top of the resin. Fifteen μl of the complex (ormore if counts were low) were added to the top of the resin. This wasfollowed by 2 ml of 4:1 (V:V) isotonic saline:conc. ammonium hydroxidesolution as an eluent which was allowed to drip into a counting tube.This was also pressure eluted to the top of the resin. An additional 2ml of the eluent were added and the column pressure eluted to remove allliquid. The dried resin was then placed in a third counting tube and thethree tubes counted on a NaI well counter using a Canberra multichannelanalyzer linked to a computer. The percent complex was determined bydividing the number of counts in the two elutions by the total counts inthe elutions plus the column, all times 100. By this method, uncomplexedyttrium was retained on the column.

Samarium Complex Preparation/% Complex Determination:

Samarium complexes were formed as described previously for yttriumcomplexes except that 0.0003M samarium was prepared by dissolution ofSm₂ 0₃ (348.7 g mole) in 0.1M hydrochloric acid. Radioactive Sm-153 wasobtained as a 0.0003M solution in 0.1M hydrochloric acid from theUniversity of Missouri Research Reactor, Columbia, Mo. Percent complexdetermination was made in the same manner as for the yttrium complex.Results are summarized in Table II.

                  TABLE II                                                        ______________________________________                                        Complexation Data                                                             Complex                                                                       Example   Compound      % Complex (10:1)                                      No.       of Ex. No.    Y       Sm                                            ______________________________________                                        16        1             98                                                    17        2             >99                                                   18        2                     >99                                           19        3             >99                                                   20        4             >99                                                   21        5             99                                                    22        6                     >99                                           23        7                       96**                                        24        8                      >99**                                        25        9                     >99                                           26        10            98                                                    27        11            99                                                    28        11             98*                                                  29        12            99                                                    30        12                     98                                           31        12             98*                                                  32        12                      98*                                         33        15            99                                                    ______________________________________                                         *Ligand/metal ratio was about 1:1;                                            **Ligand/metal ratio was about 50:1.                                     

Examples I-XV and Comparative Examples A-F IN VIVO SCREENING OFBIFUNCTIONAL CHELATES.

The stability of certain rare earth chelates has been correlated within-vivo testing in animals. For example, Rosoff, et al. in theInternational Journal of Applied Radiation and Isotopes, 14, 129-135(1963), report on the distribution of radioactive rare earth chelates inmice for certain aminocarboxylic acids. The correlation found was thatin vivo "the competition between the chelating agent and bodyconstituents (inorganic and organic) for the rare-earth ion, determinesits deposition and excretion." The strong rare-earth chelates arebelieved to dissociate very little and be excreted, while the weak andintermediate strength chelates dissociate more readily and thus aredeposited in organs such as the liver. However, concentration ofradionuclide in the liver is not always due to weak complex formation,but in some cases is due to the affinity that the metal chelate has forthe liver (see Comparative Examples A & B in Table III). Compounds have,in fact, been prepared and utilized for the evaluation of liver function(Fritzberg, Alan R., Radiopharamceuticals: Progress and ClinicalPerspectives A, (1986); U.S. Pat. Nos. 4,088,747 and 4,091,088 (Hunt etal.)

The biodistribution of several of the samarium and/or yttrium chelatesdisclosed herein was determined and the percent dose in the liver usedas an in vivo screening procedure to qualitatively estimate thestability of the chelates. Chelates of NTA and EDTA are included forcomparison. Also samarium was injected as samarium chloride inunchelated form.

Sprague-Dawley rats weighing from 150 to 200 g were purchased fromCharles River Laboratories. These animals were placed in cages and fedwater and food ad libitum. The animals were acclimated for at least fivedays before use. Prior to injection of complex, the animals were placedunder a heat lamp (15 to 30 minutes) to dilate the tail vein. Then, theanimal was placed in a restraining cage, the tail cleaned with analcohol swipe, and the animal injected (50 to 200 μl) via the tail vein.After injection, the animal was placed in another cage for two hoursafter which time the animal was sacrificed by cervical dislocation. Theanimal was then dissected, the parts rinsed with deionized H₂ O, patteddry, and weighed into a tared counting vial. Whatever size of injectionwas made, at least three standards of the same material were preparedand counted with the animal parts. Percent of dose is the number ofcounts in the organ divided by the number of counts in the standardtimes 100 (see Table III).

                  TABLE III                                                       ______________________________________                                        Biodistribution Data                                                          Biology     Compound           % Injected                                     Example     of Ex.             Dose in                                        No.         No.*       Metal   Liver                                          ______________________________________                                        I           1          Y       0.87                                           II          2          Y       0.22                                           III         3          Y       0.22                                           IV          4          Y       1.4                                            V           5          Y       0.38                                           VI          6          Sm      0.38                                           VII         9          Sm      2.8                                            VIII (10)   10         Sm      1.3                                            VIII (300)  10         Sm      0.12                                           VIII (10)   10         Y       0.39                                           VIII (300)  10         Ho      0.26                                           IX          11         Y       0.22                                           X           11         Sm      0.33                                           XI          12         Y       0.28                                           XII         12         Y       0.18                                           XIII        12         Sm      0.35                                           XIV         12         Sm      0.26                                           XV          15         Y       0.37                                           (A)         U (comp)   Sm      12                                             (B)         X (comp)   Sm      24                                             (C)         EDTA       Sm      8.4                                            (D)         EDTA       Sm      4.4                                            (E)         NTA        Sm      8.6                                            (F)         SmCl.sub.3 Sm      39                                             ______________________________________                                         *Complexes were prepared at ligand/metal ratios of 10:1 for Examples 1-XI     XIII, and XV; at 1:1 for Example XII & XIV; at 5:1 for Example C; and at      about 300:1 for Examples D and E.                                        

EXAMPLES XVI & XVII.

The 1:1 complex of yttrium with1-(p-aminobenzyl)diethylenetriaminepentaacetic acid (ABDTPA), a wellknown bifunctional chelant used in the literature, and with the ligandof Example 2 (Now Ex. XVI) and Example 12 (now Ex. XVII) were preparedusing the techniques described earlier. Several 100 microliter aliquotswere then withdrawn into separate centrifuge tubes. Excess metal wasadded such that the total volume change is minimized and the time noted.One-half hour after metal addition, the percent complex was determinedby the Sephadex™ C-25 method and this was compared to the originalamount of complex. The percent complex versus added metal gives anindication as to the lability of the ligand-metal complex. Results aregiven below and are compared to the EDTA-yttrium complex.

                  TABLE IV                                                        ______________________________________                                        Complex Study                                                                 Metal/Ligand                                                                           % Complex                                                            Molar Ratio                                                                            Ex. XVI   Ex. XVII  ABDTPA   EDTA                                    ______________________________________                                         1       99        95        97       98                                       10      94        93        95       86                                      100      84        90        92       78                                      250      --        90        87       48                                      500      75        79        70       16                                      ______________________________________                                    

EXAMPLE XVIII.

A 0.18M/L solution of 1-(p-aminobenzyl)diethylenetriaminepentaaceticacid (ABDTPA) and an identical 0.18M/L solution of the ligand of Example12 were prepared in 0.5M sodium acetate buffer at pH 6.5. The solutionswere then treated with 1.5 equivalents of yttrium-90 as 0.03M/L yttriumchloride. The pH of the resulting complex was 5-6. Excess Y-90 wasremoved by passing the complex through a one ml bed volume of Chelex™resin (Bio-Rad Laboratories). The concentration of the complex in thispurified form was 0.0013M. An appropriate amount of the solution wasadded to 1.7×10⁻⁹ moles of aldehyde containing CC-46 monoclonal antibodyto give a 40:1 ratio of complex to antibody. After one hour exposure a236 molar excess (over antibody) of NaCNBH₃ was added and the solutionsallowed to set for approximately one hour. After this time the antibody(and any covalently attached complex) was separated from unbound complexby Sephadex™ G-25 gel filtration. This procedure gave an average of 5.0complexes per antibody for1-(p-aminobenzyl)diethylenetriaminepentaacetic acid and an average of5.4 complexes per antibody for the ligand of Example 12.

EXAMPLE XIX.

In order to demonstrate the inertness of the antibody-complex conjugatesof Example XVIII, the conjugates were challenged with an excess ofdiethylenetriaminepentaacetic acid (DTPA) in the following manner. Thepurified antibody-complexes were added to HEPES buffer(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) at pH 7.4 andtreated with an appropriate amount of 0.1M DTPA solution (pH 7.4) toensure a 1000 fold molar excess of DTPA over complex attached toantibody. After one hour an aliquot was removed and the antibody-complexconjugates were separated from low molecular weight substances usinggel-filtration. The results indicate that the ABDTPA system lost over98% of the yttrium while the system using the ligand of Example 12 lostapproximately 39% of the yttrium.

EXAMPLE XX. PREPARATION OF 2,6-BIS{[(2-{BIS(CARBOXYMETHYL)]AMINO}ETHYL)(CARBOXYMETHYL)]AMINOMETHYL}-4-(AMINO)PHENOL, SAMARIUM COMPLEX

A samarium solution was prepared by combining in a 1 ml vial radioactive¹⁵³ Sm (200 μof a 3×10⁻⁴ M solution in 0.1M hydrochloric acid, 6×10⁻⁵mmole) and "cold" SmCl₃.6H₂ O (4.8 mg, 1.31×10⁻² mmole). This solutionwas added to 2,6-bis{[(2-{[bis(carboxymethyl)]amino}ethyl)(carboxymethyl)]aminomethyl}-4-(acetamido)phenol (3.2 mg, 5.31×10⁻³mmole), prepared by the procedure of Example 11. The pH was thenadjusted to 7 by the addition of sodium hydroxide (40 ul of a 1.0Msolution). The percent complex was determined to be 68% using theSephadex™ C-25 method.

The above complex was purified by anion exchange chromatography(Q-Sepharose™, 1.5 cm ×21 cm, 0 to 1M NaCl over 30 min, 2 ml/min,detection at 285 nm). Complex-containing fractions (1 ml each, 6 mltotal) were combined and the percent complex was determined to be 95%.

EXAMPLE XXI. CONJUGATION OF 2,6-BIS{[(2-{BIS(CARBOXYMETHYL)]AMINO}ETHYL)(CARBOXYMETHYL) ]AMINOMETHYL}-4-(AMINO)PHENOL, SAMARIUM COMPLEX TO CC-46MONOCLONAL ANTIBODY

Sodium bicarbonate (60 mg, 7.14×10⁻¹ mmole) was placed in a one dramglass vial and the complex solution from Example XX was added (1 ml,about 8.8×10⁻⁴ mmole). Thiophosgene (10 μl, 1.31×10⁻¹ mmole) inchloroform (1 ml) was added and the vial was sealed. The mixture wasshaken for 15 minutes, after which the aqueous layer was washed twicewith chloroform (1 ml portions). Percent complex was checked and foundto be 96%.

The above isothiocyanate Sm complex (100 μl, about 8.8×10⁻⁵ mmole) wascombined with CC-46 monoclonal antibody (100 μl of an 8 mg/ml solution,about 5.3×10⁻⁶ mmole) and allowed to stand for 24 hours. The amount ofcomplex conjugated to antibody was determined to be 46% by sizeexclusion chromatography.

EXAMPLE XXII. PREPARATION OF 2-[BIS(2-{[(BIS(CARBOXYMETHYL)]AMINO}ETHYL)AMINO]-2-[5-AMINO-2-(CARBOXYMETHYLOXY) PHENYL]ETHANOIC ACIDAND 2-[{2-[(2{[BIS(CARBOXYMETHYL)]AMINO}ETHYL)(CARBOXYMETHYL)AMINO]ETHYL}(CARBOXYMETHYL)AMINO]-2-[5-AMINO-2-(CARBOXYMETHYLOXY)PHENYL]ETHANOICACID, SAMARIUM COMPLEX

A solution of the ligands from Example 7 was prepared by dissolving 266mg of the lyophilized solid in 1 ml of water. A 33.85 μl aliquot of thissolution was treated with 1 ml of 3×10⁻⁴ M SmCl₃ in 0.1N hydrochloricacid containing a tracer amount of radioactive ¹⁵³ Sm. The pH of thecomplex solution was adjusted to about 13 using 50 weight % sodiumhydroxide and then adjusted to about pH 7.5 using 1.ON hydrochloricacid. The percent Sm that was complexed was determined as described inExamples 16 through 33 and was found to be 100%.

The inertness of the complex was demonstrated by placing two 500 μlaliquots of the complex solution in separate vials. One portion wastreated with 1-2 μl portions of 0.1N hydrochloric acid until the pH waslowered and the other portion was treated with 0.1N sodium hydroxide tobring the pH up. The complexes were allowed to set for 5-10 minutes ateach pH change, then they were sampled to determine the percentcomplexation at that pH by the method described for Examples 16 through33. The results are shown in the following table.

                  TABLE V                                                         ______________________________________                                               pH   % Complexed                                                       ______________________________________                                               1     98                                                                      2    100                                                                      3    100                                                                      4    100                                                                      5    100                                                                      7    100                                                                      9    100                                                                      11   100                                                                      13   100                                                               ______________________________________                                    

EXAMPLE XXIII. PREPARATION OF 2-[{2-[(2{[BIS(CARBOXYMETHYL)]AMINO}ETHYL) (CARBOXYMETHYL) AMINO]ETHYL}(CARBOXYMETHYL) AMINO]-2-[5-AMINO-2-(CARBOXYMETHYLOXY)PHENYL]ETHANOICACID, SAMARIUM COMPLEX

A solution of the ligand from Example 8 was prepared by dissolving 13.9mg of the brownish solid in 772 μl of water. A complex was prepared bydissolving 500 of this ligand solution in 1 ml of 3×10⁻⁴ M SmCl₃(containing 0.1N hydrochloric acid) that had been spiked withradioactive ¹⁵³ Sm. The pH of the complex solution was adjusted to about7 by the addition of 1.0N sodium hydroxide. The percent complexation wasdetermined by the method described for Examples 16 through 33 and foundto be 96 %.

The inertness of the complex was demonstrated by placing two 500 μlaliquots of the complex solution in separate vials. One portion wastreated with 1-2 μportions of 1.0N hydrochloric acid until the pH waslowered and the other portion was treated with 0.1N, 1.0N and 50 weight% sodium hydroxide to bring the pH up. The complexes were allowed to setfor about 5 minutes at each pH change, then they were sampled todetermine the percent complexation at that pH by the method describedfor Examples 16 through 33. The results are shown in the followingtable.

                  TABLE VI                                                        ______________________________________                                               pH   % Complexed                                                       ______________________________________                                               1    91                                                                       2    88                                                                       3    92                                                                       5    96                                                                       7    96                                                                       9    99                                                                       12   98                                                                       13   99                                                                ______________________________________                                    

EXAMPLES OF BIODISTRIBUTION DATA

Complexes were prepared by mixing a solution of ligand and metal thenadjusting the pH to 7-8. The amount of metal that was complexed toligand was determined by cation exchange chromatography. Free metal wasretained by the resin, metal in the form of a complex was not.

One-hundred μl of the complexes were injected into the tail vein ofthree Sprague Dawley rats. Two hours after injection, the rats werekilled by cervical dislocation and samples of tissues were taken. Thetissues were weighed and the amount of radiation in each tissuedetermined by measuring the number of counts using a NaI well counterand comparing them to standards. The % dose in blood was determinedassuming that the weight of blood was 6.5% of the animal weight. Musclewas calculated using 46% of body weight. The amount in bone was 25 timesthe % dose in a femur. The examples below differ in the ligand, amountof ligand and amount of metal used. Non-radioactive metal was used toobtain the desired ligand to metal ratios and tracer radioactive metalwas used to obtain the biodistribution.

EXAMPLE XXIV.

The ligand of Example 10 was mixed with a Sm-153 solution. Theconcentration of Sm was 3×10⁻⁴ M and the ligand was used with a 300times molar excess. The biodistribution showed 52.7% in the bone, 0.12%in the liver, 0.005% in the spleen, 0.23% in the muscle, and 0.05% inthe blood.

EXAMPLE XXV.

The ligand of Example 10 was complexed to Ho-166. The concentration ofHo was 3X10-4M and the formulation contained 300 times molar excessligand. The biodistribution showed 52.9% in the bone, 0.26% in theliver, 0.007% in the spleen, 1.1% in the muscle, and 0.09% in the blood.

EXAMPLE XXVI.

The ligand of Example 10 was complexed to Sm-153 using a concentrationof Sm of 3×10⁻⁴ M and 10 times molar excess ligand. The biodistributionshowed 48.5% in the bone, 1.3% in the liver, 0.01% in the spleen, 0.73%in the muscle and 0. 18% in the blood.

EXAMPLE XXVII.

A rabbit was injected in the same manner as the rats with a formulationhaving Y-90 with Y at 3×10⁻⁴ M and the ligand of Example 10 at 10 timesmolar excess. The activity was found to concentrate in bone (59%) withliver (1.1%), spleen (0.19%), muscle (1.5%) and blood (0.68%) showingminimal uptake.

EXAMPLE XXVIII.

The ligand of Example 1 was complexed using Y-90 as a tracer. The Yconcentration was 3×10⁻⁴ M and the ligand was added in a 10 times molarexcess. The rat biodistribution (average of two rats) showed 56.1% inthe bone, 0.87% in the liver, 0.03% in the spleen, 0.78% in the muscle,and 0.57% in the blood.

EXAMPLE XXIX

A dog was presented with an osteosarcoma in the right proximal humerusand walked with significant lameness. A complex was prepared using theligand of Example 10 with a Sm-153 solution. The concentration of Sm was3×10⁻⁴ M and the ligand was used with a 300 times molar excess. Thespecific activity of the Sm-153 was 30 mCi/ml. The dog was given an I.V.injection of this complex containing 0.95 mCi of Sm-153 per Kg bodyweight of the dog. One week after injection the dog's gait wasnoticeably improved.

    TABLE 1      GENERIC STRUCTURE      ##STR7##      (I) Example No. Z X R.sub.5 n R.sub.1 R.sub.2 R.sub.3 R.sub.4 B                 1 NHCOCH.sub.3 H H 0 -- -- H COOH      ##STR8##       2 NHCOCH.sub.3 CH.sub.2      COOH H 0 -- -- H COOH            ##STR9##       3 NH.sub.2 CH.sub.2      COOH H 0 -- -- H COOH            ##STR10##      4 NHCOCH.sub.3 H H 0 -- -- H COOH            ##STR11##      5 NH.sub.2 H H 0 -- -- H COOH            ##STR12##       6 NHCOCH.sub.3 CH.sub.2 CO.sub.2      H H 0 -- -- H COOH            ##STR13##      ##STR14##       7 NH.sub.2 CH.sub.2      COOH H 0 -- -- H COOH            ##STR15##      ##STR16##       8 NH.sub.2 CH.sub.2      COOH H 0 -- -- H COOH            ##STR17##       9 NHCOCH.sub.3 CH.sub.2 CO.sub.2      H H 0 -- -- H COOH            ##STR18##      ##STR19##       10       NHCOCH.sub.3 H            ##STR20##      0 -- -- H COOH      ##STR21##       11       NHCOCH.sub.3 H            ##STR22##      0 -- -- H H      ##STR23##       12       NH.sub.2 H            ##STR24##      0 -- -- H H      ##STR25##       13       NCS H            ##STR26##      0 -- -- H H      ##STR27##       14       NHCOCH.sub.3 H H 0 -- -- H H            ##STR28##      15 NHCOCH.sub.3 H            ##STR29##      0 -- -- H H      ##STR30##

What we claim is:
 1. A complex comprising a chelant possessing ortholigating functionality having the formula ##STR31## wherein Z' isselected from the group consisting of hydrogen, NH₂, NO₂, NHC(O)CH₃ orN(R')₂, where R' is hydrogen and C₁ -C₃ alkyl;X is selected from thegroup consisting of hydrogen, C₁ -C₃ alkyl and CR₃ R₄ COOH; R₃ and R₄are independently selected from the group consisting of hydrogen, C₁ -C₃alkyl and COOH; R'₁ and R'₂ each are independently selected from thegroup consisting of hydrogen and COOH, with the proviso that at leastone is COOH; R'₃, R'4, R'₅ and R'₆ are independently selected from thegroup consisting of hydrogen and CR₃ R₄ COOH, with the proviso that atleast three are CR₃ R₄ COOH; or a pharmaceutically acceptable saltthereof; and complexed with a radionuclide metal ion selected from thegroup consisting of ¹⁵³ Sm, ¹⁶⁶ Ho, ⁹⁰ y, ¹⁴⁹ pm, ¹⁵⁹ Gd, ⁰ La, ¹⁷⁷ Lu,¹⁷⁵ Yb, ⁴⁷ Sc and ¹⁴² Pr.
 2. The chelant of claim 1 wherein R'₁ and R'₂are COOH, R'₃, R'₄, R'₅ and R'₆ are CH₂ COOH.
 3. The chelant of claim 1wherein Z' is NHC(O)CH₃ and X is hydrogen.
 4. A complex of claim 1wherein the metal ion is ¹⁵³ Sm, ¹⁶⁶ Ho, ⁹⁰ y, ¹⁵⁹ Gd, ¹⁷⁷ Lu or ¹⁷⁵ Yb.5. A complex of claim 27 wherein the metal ion is ¹⁵³ Sm, ¹⁶⁶ Ho, ⁹⁰ y,¹⁵⁹ Gd or ¹⁷⁷ Lu.
 6. A therapeutically effective formulation comprisingthe complex of claim 1 or a physiologically acceptable salt thereof, anda physiologically acceptable carrier.
 7. A method for the therapeutictreatment of an animal having one or more calcific tumors whichcomprises administering to said animal a therapeutically effectiveamount of a formulation of claim
 6. 8. A method for the therapeutictreatment of an animal having bone pain which comprises administering tosaid animal a therapeutically effective amount of a formulation of claim6.